The long-range goal of the proposed studies is to identify the mechanisms of cell fate specification and gastrulation during vertebrate development. Our focus is on the Nodal TGFbeta signaling pathway, which plays fundamental roles during vertebrate development. Previous studies have revealed that (A) EGF-CFC proteins are required for Nodal signaling and the development of mesoderm, endoderm and left-right asymmetry; (B) Lefty TGFbeta proteins act as feedback inhibitors of Nodal signaling; (C) Nodal signals can act at a distance as long-range morphogens. The proposed research addresses three fundamental questions: (A) What is the molecular basis of the interactions between TGFbeta signals, EGF-CFC proteins and TGFbeta receptors? (B) How is the range of Nodal signaling regulated? (C) Which genes mediate the effects of Nodal signaling during gastrulation? The proposal has three aims: (1) To determine how EGF-CFC proteins interact with TGFbeta receptors and the TGFbeta signals Nodal, Vg1, GDF1, Activin and Lefty, we will use reporter assays and biochemical analysis in Xenopus. (2) To determine how Lefty, EGF-CFC proteins and TGFbeta receptors regulate the range of Nodal signaling, we will use reporter gene assays and study the in vivo distribution of GFP-TGFbeta fusion proteins in zebrafish embryos. (3) We have identified three novel genes regulated by Nodal signaling during zebrafish gastrulation. To determine their role during mesoderm and endoderm development and gastrulation, we will use misexpression, morpholino-mediated knock-down and dominant negative approaches. Mutations in EGF-CFC genes in humans are associated with forebrain and left-right defects. The proposed studies will thus help to provide the necessary context for understanding these developmental abnormalities.